JPWO2019203255A1 - 再構成癌組織を用いた薬剤評価方法 - Google Patents
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Abstract
Description
1)単剤投与による膵癌治療法
・JAMA, 297, 267-77, 2007.(非特許文献1): 膵癌に対するGemcitabineの単独療法の報告である。
・特許第5909767号(特許文献1): 膵癌などのForkhead box M1(FOXM1) を高発現する癌に対するワクチンとして有効な、新規ペプチドの報告である。
・特許第5904552号(特許文献2): 膵癌で発現するインターロイキン6(IL-6)の阻害剤を用いた治療の報告である。
・特開2015-221793(特許文献3): ヒトTROP2の阻害抗体を用いた治療法の報告である。
・特開2017-48170(特許文献4): クローディン18に対する抗体を用いて膵癌等の増殖抑制を図るものである。
・特開2017-141163(特許文献5): 膵癌で発現が亢進するリボソームタンパク質であるRPL29を標的として治療を図る方法である。
2)併用投与による膵癌治療法
・JAMA 304, 1073-81, 2010(非特許文献2): Gemcitabineとフルオロウラシルの併用療法であるが、標準治療には採用されていない。
・Lanccet, 388-248-57, 2016.(非特許文献3): GemcitabineとTS-1の併用療法の報告である。
(1)癌微小環境を再構成する組織中の癌細胞及び/又は間質細胞において、抗癌剤の投与前と投与後のEMT関連分子の発現レベルを測定し、抗癌剤の投与前よりも投与後の発現レベルが上昇しているEMT関連分子を癌の治療抵抗性に関与していると判定する、癌の治療抵抗性に関与する分子のスクリーニング方法。
(2)癌微小環境を再構成する組織中の癌細胞及び/又は間質細胞において、抗癌剤の投与前と投与後のEMT関連分子の発現レベルを測定し、抗癌剤の投与前よりも投与後の発現レベルが上昇しているEMT関連分子を癌の治療抵抗性に関与していると判定し、そのEMT関連分子の機能を阻害できる物質を癌の治療抵抗性に有効な薬剤であると判定する、癌の治療抵抗性に有効な薬剤のスクリーニング方法。
(3)癌の治療抵抗性に有効な薬剤が癌の再発の治療及び/又は予防に有効な薬剤である(2)記載の方法。
(4)抗癌剤とNOTCH3シグナルを標的とする阻害薬とを併用する、再発腫瘍の治療及び/又は予防薬。
本明細書は、本願の優先権の基礎である日本国特許出願、特願2018‐80585の明細書および/または図面に記載される内容を包含する。
a: 間葉系細胞のマーカー(α-SMA)を指標とした免疫染色による定量(図7参照)。原発巣(61%程度)に対して、後述の実施例で作製した膵癌オルガノイド(10:7:20)は59%程度であった。本発明の癌オルガノイドは、この定量方法により、1〜1000%であるとよく、好ましくは、10〜500%であり、より好ましくは、10〜300%である(免疫染色による陽性率)。
b: 間質内の細胞外マトリックス(膵癌の場合は、ヒアルロン酸・コラーゲンなど)の定量(コラーゲンについては、シリウス赤染色による定性解析、シリウス赤染色後の偏光顕微鏡像解析による定量解析が可能である(図8参照)。後述の実施例では、原発巣(74%程度)に対して、膵癌オルガノイド(10:7:20)は44%程度であった。本発明の癌オルガノイドは、この定量方法により、1〜1000%であるとよく、好ましくは、10〜500%であり、より好ましくは、10〜300%である。
c: 間質内にヒアルロン酸やコラーゲンが蓄積すると、組織の硬度が増加する。組織の硬さを指標として判断することも可能である。
LY3039478:4,4,4-trifluoro-N-[(1S)-1-{[(10S)-8-(2-hydroxyethyl)-9-oxo-6,8-diazatricyclo[9.4.0.0^{2,7}]pentadeca-1(15),2,4,6,11,13-hexaen-10-yl]carbamoyl}ethyl]butanamide
MK-0752:3-[(1s,4r)-4-(4-chlorobenzenesulfonyl)-4-(2,5-difluorophenyl)cyclohexyl]propanoic acid
PF-03084014:(2S)-2-{[(2S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl]amino}-N-(1-{1-[(2,2-dimethylpropyl)amino]-2-methylpropan-2-yl}-1H-imidazol-4-yl)pentanamide
〔実施例1〕
プライマリヒト膵癌オルガノイドを対象とした抗癌剤(ゲムシタビン)の薬剤感受性評価(図1)
プライマリヒト膵癌細胞(膵癌患者から切除した組織から単離した細胞を増やしたもの)にルシフェラーゼ遺伝子を導入した後、プライマリ膵癌細胞の基本培地とEGM培地 (Lonza) の1:1混合液を使用して、ストロマ細胞(ヒト間葉系幹細胞(hMSC)、ヒト臍帯静脈内皮細胞(HUVEC)いずれもLonza社)との三次元共培養を行い(プライマリヒト膵癌細胞:hMSC:HUVEC=10:7:20)、豊富な間質を持つプライマリヒト膵癌オルガノイドを再構成した。再構成されたプライマリヒト膵癌オルガノイドに各濃度の抗癌剤(ゲムシタビン)を添加し、それぞれの薬剤感受性を評価した。豊富な間質を持つプライマリヒト膵癌オルガノイド(図中赤線●)は、プライマリヒト膵癌細胞のみで構成される群(図中青線■)と比べて、抗癌剤の抵抗性が亢進している。
間質豊富なプライマリヒト膵癌オルガノイドは、抗癌剤に対して高い治療抵抗性を示した。癌間質を介した膵癌細胞の治療抵抗性亢進についてのメカニズムを明らかにするため、間質豊富なプライマリヒト膵癌オルガノイドと膵癌細胞との間における遺伝子発現および蛋白質発現を比較した。間質豊富なプライマリヒト膵癌オルガノイドより分離した膵癌細胞と膵癌細胞単独培養後の膵癌細胞の間の遺伝子発現状態をマイクロアレイ解析により比較したところ、EMT関連因子、癌の悪性度への関連が報告された因子、腫瘍形成に関与することが報告された分子が多数抽出された。また、間質豊富なプライマリヒト膵癌オルガノイドと膵癌細胞の培養上清を対象としたプロテオーム解析(セクレトーム解析)を行ったところ、EMT関連分子(NOTCH3等)が複数抽出された。
間質豊富なプライマリヒト膵癌オルガノイドとプライマリヒト膵癌細胞の間で遺伝子発現状態を比較するため、各々のtotalRNAを用いてマイクロアレイデータ(Agilent社)を取得し、得られたアレイデータを用いてGene Set Enrichment Analysis (GSEA)解析を行った。双方のサンプル間で変動する遺伝子群がいずれの遺伝子セットに該当するかを検討したところ、膵癌オルガノイドは膵癌細胞のみと比べてEMT遺伝子セットが遺伝子発現データ中で有意に変動することが確認された。図中左側が膵癌オルガノイドの発現遺伝子、右側が膵癌細胞の発現遺伝子を示し、EMT関連遺伝子の検出頻度が左側で優位に高いことが確認された。
固形癌では癌細胞が治療抵抗性を獲得する過程において、癌細胞の特性が上皮様から間質様に変化することが知られている(上皮間葉転換:Epithelial-Mesenchymal Transition : EMT)。多くの癌においてEMTへの移行期(Partial EMT)より癌細胞の薬剤抵抗性が亢進することが報告されている。
前述の方法で作製した膵癌オルガノイドあるいは、膵癌シストをDMEM/F12培地で洗浄したのち、ピペットを用いて細かく分散して、DMEM/F12培地とマトリゲルの混合液(1:1混合液)中に懸濁し、200ulプラスティックチップを用いて回収し、重度免疫不全マウス(NSGマウス、チャールズリバー)の背部皮下に移植してゼノグラフトを作製した(膵癌オルガノイド由来ゼノグラフト、膵癌シスト由来ゼノグラフト)。
膵癌オルガノイドから形成されたゼノグラフト(Organoid)でEMTマーカー(ZEB2, pSMAD2/3, N-CADHERIN)の発現を評価したところ(免疫染色法)、CK7を発現する膵癌細胞においてEMTマーカーの発現亢進が生じることが確認された。また、膵癌オルガノイドから形成されたゼノグラフトは、ゲムシタビン治療を施した後(ゼノグラフトのサイズが100mm3に達した後、ゲムシタビン(90mg/kg)を3日毎に腹腔内投与した。)においても、EMT関連分子の発現が亢進していることが確認された(図5は、治療開始後30日目の組織を示す。)。
膵癌オルガノイドから形成されたゼノグラフト(Org)および膵癌シストから形成されたゼノグラフト(Cyst)でNOTCH分子群の発現を評価した(免疫染色法)。間質豊富な膵癌オルガノイドで発現が亢進し、抗癌剤投与に発現が持続する分子としてNOTCH3が同定された。NOTCH3の発現は膵癌細胞と膵癌間質細胞の双方で亢進することが確認された。図6Aにゲムシタビン投与前後での膵癌オルガノイド由来ゼノグラフトあるいは膵癌シスト由来ゼノグラフトでのNOTCH分子群の発現状態を示す。図Bに免疫染色結果の定量結果を示す。サイトケラチン7(CK7)を発現する膵癌細胞におけるNOTCH分子群の発現結果を示す。図4では膵癌細胞におけるNOTCH下流シグナルの発現解析結果を示す。図Dに膵癌の原発巣と間質を有する膵癌オルガノイドでのNOTCH3の発現状態を示す。Smooth muscle actin(SMA)は間葉系細胞のマーカーとして使用した。図6Eは術後にゲムシタビン(GEM)あるいは、TS-1治療を行った患者の生存期間を示す。手術時の膵癌組織における癌細胞でのNOTCH3発現、および間質細胞でのNOTCH3発現に基づき生存頻度をプロットした。
プライマリヒト膵癌オルガノイドに由来するゼノグラフトに膵癌治療薬(ゲムシタビン)を投与し(ゲムシタビン(90mg/kg)を3日毎に腹腔内投与した。)、治療後に残存する膵癌細胞の特性を検討した。図7は、治療開始後30日目の組織を示す。治療後に残存した膵癌細胞はNOTCH3の発現が高く(免疫染色法)、また、細胞増殖が亢進していることが確認された。膵癌細胞におけるNOTCH3の発現を3段階に分類し(Ki67染色像(核)を基準に細胞を分類し、細胞周囲のNOTCH3の発現の強さで分類した。全体の発現量を元に、NOTCH3発現なし、弱染色、強染色の三段階で評価した。)、それぞれの群でKi67を発現し、増殖性を示す膵癌細胞の頻度を検討した。その結果、抗癌剤治療後に残存するNOTCH3強陽性細胞では、増殖している細胞の割合が高いことが確認された。NOTCH3陽性細胞は、抗癌剤に高い薬剤抵抗性を示すことが確認された。
NOTCH3阻害作用を有する阻害剤(LY 3039478 (10 mg / kg, Eli Lily))がヒト膵癌の治療に有効か否かを検討した。重度免疫不全マウス(NSGマウス、チャールズリバー)背部皮下にプライマリヒト膵癌オルガノイドを移植し、ゼノグラフトが一定のサイズ(100mm3)に達した時点から治療を開始した。膵癌治療薬であるゲムシタビンの投与群、NOTCH3阻害剤の投与群、ゲムシタビンとNOTCH3阻害剤の双方の投与群(併用群)、未治療群について腫瘍サイズの変化を検討した。NOTCH3阻害剤投与群では、ゲムシタビン治療群に近い治療効果が確認されるものの、膵癌組織の増大を完全に抑制することは困難であった。一方で、NOTCH3阻害剤とゲムシタビンの併用治療群では、膵癌組織の増大が強く抑制され、併用治療の有効性が明らかとなった。図中右に各治療後の膵癌ゼノグラフトのマクロ像およびHE染色像を示す。
膵癌は癌の進展が早く、再発・転移が高頻度に生じる。再発した膵癌に対する有効な治療法は確立されておらず、新たな治療法の確立が必須である。再発膵癌の治療におけるNOTCH3阻害剤の有効性を検証するため、プライマリヒト膵癌オルガノイドを重度免疫不全マウス(NSGマウス、チャールズリバー)に移植し、腫瘍サイズが一定(100mm3)に達した時点からゲムシタビン投与を施した。投与後、腫瘍サイズが減少した後、ゲムシタビン投与を中断すると膵癌の増殖が亢進することが確認された。本手法は、膵癌の再発を再現するモデルとして有益であることが確認された。
また、同再発モデルの治療にNOTCH3が有益か否かを検討した。腫瘍サイズが一定(100mm3)に達した時点からNOTCH3阻害剤、ゲムシタビン投与を行い、腫瘍サイズの変化を検討したところ、NOTCH3阻害剤あるいはゲムシタビンのいずれかを投与することで腫瘍サイズの増加を抑制できることが確認された。さらに、NOTCH3阻害剤とゲムシタビンの併用治療の効果を検討したところ、併用群では再発後の腫瘍サイズが大きく減少することが確認された。膵癌の再発にNOTCH3阻害剤とゲムシタビンの併用治療が有効であることが確認された。図中右に各治療後の膵癌ゼノグラフトのマクロ像およびHE染色像を示す。
膵癌オルガノイド由来ゼノグラフトについてNOTCH3阻害剤、ゲムシタビン、併用治療を実施した後の間質量をシリウスレッド染色により評価した。図中左側上段に治療後のゼノグラフトのHE染色像、下段にシリウスレッド染色像を示す。NOTCH3阻害剤を用いて治療すると、膵癌組織中のシリウスレッド陽性領域が減少することが明らかとなった(右図)。また、図中央は、細胞核の染色により膵管部位での細胞の重層化の程度を評価し、平均値で示した。グラフ上の点は個々の膵管部位を示す。
NOTCH3治療後の膵癌組織においてEMT関連分子の発現を評価したところ(免疫染色)、NOTCH3阻害剤投与群においてEMT関連分子を発現する細胞が減少することが確認された(左図)。それぞれの治療群においてEMT関連分子・細胞増殖マーカー・幹細胞マーカー・細胞外マトリクス・繊維化マーカーの発現を評価したところ(免疫染色)、NOTCH3阻害剤とゲムシタビン併用群ではこれらの分子が抑制されることが明らかとなった。一方、NOTCH3阻害剤とゲムシタビン併用群では、カスパーゼ活性などのアポトーシスマーカーの発現が亢進することが確認された(右図)。
本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。
Claims (4)
- 癌微小環境を再構成する組織中の癌細胞及び/又は間質細胞において、抗癌剤の投与前と投与後のEMT関連分子の発現レベルを測定し、抗癌剤の投与前よりも投与後の発現レベルが上昇しているEMT関連分子を癌の治療抵抗性に関与していると判定する、癌の治療抵抗性に関与する分子のスクリーニング方法。
- 癌微小環境を再構成する組織中の癌細胞及び/又は間質細胞において、抗癌剤の投与前と投与後のEMT関連分子の発現レベルを測定し、抗癌剤の投与前よりも投与後の発現レベルが上昇しているEMT関連分子を癌の治療抵抗性に関与していると判定し、そのEMT関連分子の機能を阻害できる物質を癌の治療抵抗性に有効な薬剤であると判定する、癌の治療抵抗性に有効な薬剤のスクリーニング方法。
- 癌の治療抵抗性に有効な薬剤が癌の再発の治療及び/又は予防に有効な薬剤である請求項2記載の方法。
- 抗癌剤とNOTCH3シグナルを標的とする阻害薬とを併用する、再発腫瘍の治療及び/又は予防薬。
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